Scrapie is a naturally occurring spongiform encephalopathy of sheep and goats which causes clinical and pathological changes similar to those of Creutzfeld-Jakob and Kuru diseases of man. A unique protein called prion protein (PrP) has been found to be a major component of purified samples containing scrapie infectivity and is believed by some people to be the infectious agent. It is just as possible though that the protein accumulates as a secondary by- product of the disease in vivo. To discriminate between these possibilities we studied PrP biosynthesis in vitro in scrapie infected and uninfected neuroblastoma cells using pulse-chase labelling experiments. No differences in PrP biosynthesis were found, and this suggested a secondary role for PrP in scrapie pathogenesis. Because animal tissues have not provided satisfactory substrates for the biochemical analysis or isolation of the scrapie agent, we have continued to develop and improve an in vitro scrapie infected cell culture system which we described before. Scrapie infected cultures were derived from single positive cells and contained 50- 100 fold more agent than was found in uncloned cultures. By frequency analysis nearly every cell in expanded cultures continued to contain scrapie agent. Cell dose-infectivity relationships were studied and a standard curve was developed which allowed various cultures to be compared, thus significantly reducing the numbers of animals needed to titrate various samples. These cultures will be used to test methods for agent purification.